miRNA is a single-stranded RNA having about 22 to 25 bases, and is a functional small RNA involved in cellular proliferation, differentiation, and development. The miRNA binds to a 3′ untranslated region of mRNA of a gene serving as a target to suppress translation (protein synthesis) of the target genes, thereby being involved in expression control. Hitherto, as many as hundreds of kinds of miRNAs have been identified, and further novel kinds are estimated to be discovered in the future. In recent years, it has been revealed that aberrant expression of the miRNA, for example, is involved in cancer and a variety of other diseases. Accordingly, there have been increasing needs for elucidation of target genes of the miRNA from standpoints of not only basic research but also clinical research. For example, it has been becoming clear that particular miRNAs are expressed at high levels or low levels in certain kinds of cancer cells. However, true target genes of most of the miRNAs are unknown. When cancer is developed because of high level expression of miRNA, a target gene whose protein synthesis is regulated by the miRNA may be estimated to be a gene involved in tumor suppression (tumor suppressor gene). On the other hand, when cancer is developed because of low level expression of miRNA, the target gene of the miRNA may be estimated to be a gene involved in carcinogenesis (oncogene). It is an important task to reveal true target genes of the miRNA, thereby elucidating pathogenic mechanisms of cancer and other diseases and developing molecular-targeted therapeutic drugs therefor.
The miRNA plays an important role in regulation of gene expression. The miRNA is transcribed from DNA in the form of a longer RNA as a precursor in the nucleus and synthesized as pri-miRNA, which is processed by Drosha into pre-miRNA to be transferred to the cytoplasm. The pre-miRNA is processed through action of Dicer into a double-stranded RNA of about 22 to 25 bases, one RNA strand of which is released and the remaining strand of which is incorporated into an RNA-induced silencing complex (RISC complex) to be involved as the miRNA in gene function regulation. It is known that the RISC complex contains an AGO protein. The miRNA in the RISC complex binds to a 3′ untranslated region (3′UTR) of mRNA of genes serving as targets. The binding mode of the miRNA is such that not the entirety of its sequence forms a pair but the miRNA binds mainly at a region of about 8 bases at its 5′ side (called a seed sequence) and binds in such a form as to contain a mismatch as a whole. This complicates a search for target genes of the miRNA. Some types of target gene prediction software each using a unique algorithm for the target search (miCTS, TargetScan, PicTar, MiRanda, and the like) have been produced. However, target candidate genes selected as a result of the software analysis are tremendous in number, and hence the software analysis at present is far from allowing practical narrowing down of candidate genes.
As a method of narrowing down target genes of the miRNA, there has been reported a method involving purifying mRNA bound to miRNA (Non Patent Literature 1). The method of narrowing down target genes of the miRNA disclosed in Non Patent Literature 1 involves: first inserting a FLAG-tagged AGO gene into cells through infection with a retroviral vector; further transfecting the cells with 3′ end biotinylated double-stranded miRNA through use of a transfection reagent; then lysing the cells to produce a cell extract; pulling down an AGO protein complex with anti-FALG antibody beads; next adding a large amount of a FLAG peptide to liberate the AGO protein complex from the anti-FALG antibody beads; subsequently performing an operation of pulling down a biotinylated miRNA complex (containing target gene mRNA) with streptavidin beads; and identifying the thus pulled down mRNA. However, the method is considered to have, for example, the following problems. Laboratories capable of performing the retroviral infection operation are limited. In addition, as many as two gene transfection operations are necessary, resulting in a significant drawback of a reduction in transfection efficiency into cells. Moreover, the mRNA corresponding to the target gene is degraded through the two pull down operations.